Oral delivery of protein drugs is considered a life-changing solution for patients who require regular needle injections. However, clinical translation of oral protein formulations has been hampered by inefficient penetration of drugs through the intestinal mucus and epithelial cell layer, leading to low absorption and bioavailability, and safety concerns owing to tight junction openings. Here we report a zwitterionic micelle platform featuring a virus-mimetic zwitterionic surface, a betaine side chain and an ultralow critical micelle concentration, enabling drug penetration through the mucus and efficient transporter-mediated epithelial absorption without the need for tight junction opening. This micelle platform was used to fabricate a prototype oral insulin formulation by encapsulating a freeze-dried powder of zwitterionic micelle insulin into an enteric-coated capsule. The biocompatible oral insulin formulation shows a high oral bioavailability of >40%, offers the possibility to fine tune insulin acting profiles and provides long-term safety, enabling the oral delivery of protein drugs.
Subscribe to Journal
Get full journal access for 1 year
only $14.08 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The authors declare that all data supporting the findings of this study are available within the paper and its Extended Data.
Sinha, V. et al. Oral colon-specific drug delivery of protein and peptide drugs. Crit. Rev. Ther. Drug Carrier Syst. 24, 63–92 (2007).
des Rieux, A., Fievez, V., Garinot, M., Schneider, Y.-J. & Preat, V. Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach. J. Control. Release 116, 1–27 (2006).
Muheem, A. et al. A review on the strategies for oral delivery of proteins and peptides and their clinical perspectives. Saudi Pharm. J. 24, 413–428 (2016).
Serra, L., Domenech, J. & Peppas, N. A. Drug transport mechanisms and release kinetics from molecularly designed poly(acrylic acid-g-ethylene glycol) hydrogels. Biomaterials 27, 5440–5451 (2006).
Yu, F. et al. Enteric-coated capsules filled with mono-disperse micro-particles containing PLGA-lipid-PEG nanoparticles for oral delivery of insulin. Int. J. Pharm. 484, 181–191 (2015).
Mustata, G. & Dinh, S. M. Approaches to oral drug delivery for challenging molecules. Crit. Rev. Ther. Drug Carrier Syst. 23, 111–135 (2006).
Goldberg, M. & Gomez-Orellana, I. Challenges for the oral delivery of macromolecules. Nat. Rev. Drug. Discov. 2, 289–295 (2003).
Ensign, L. M., Cone, R. & Hanes, J. Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers. Adv. Drug Deliver. Rev. 64, 557–570 (2012).
Cu, Y. & Saltzman, W. M. Mathematical modeling of molecular diffusion through mucus. Adv. Drug. Deliver. Rev. 61, 101–114 (2009).
Saltzman, W. M., Radomsky, M. L., Whaley, K. J. & Cone, R. A. Antibody diffusion in human cervical-mucus. Biophys. J. 66, 508–515 (1994).
Chilvers, M. A. & O’Callaghan, C. Local mucociliary defence mechanisms. Paediatr. Respir. Rev. 1, 27–34 (2000).
Knowles, M. R. & Boucher, R. C. Mucus clearance as a primary innate defense mechanism for mammalian airways. J. Clin. Invest. 109, 571–577 (2002).
McAuley, J. L. et al. MUC1 cell surface mucin is a critical element of the mucosal barrier to infection. J. Clin. Invest. 117, 2313–2324 (2007).
Cone, R. Barrier properties of mucus. Adv Drug Deliver. Rev. 61, 75–85 (2009).
Lai, S. K., Wang, Y. & Hanes, J. Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues. Adv. Drug Deliver. Rev. 61, 158–171 (2009).
Ensign, L. M., Schneider, C., Suk, J. S., Cone, R. & Hanes, J. Mucus penetrating nanoparticles: biophysical tool and method of drug and gene delivery. Adv. Mater. 24, 3887–3894 (2012).
Lai, S. K. et al. Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus. Proc. Natl Acad. Sci. USA 104, 1482–1487 (2007).
Maisel, K., Ensign, L. M., Reddy, M., Cone, R. & Hanes, J. Effect of surface chemistry on nanoparticle interaction with gastrointestinal mucus and distribution in the gastrointestinal tract following oral and rectal administration in the mouse. J. Control. Release 197, 48–57 (2015).
Cu, Y. & Saltzman, W. M. Controlled surface modification with poly(ethylene)glycol enhances diffusion of PLGA nanoparticles in human cervical mucus. Mol. Pharmaceut. 6, 173–181 (2009).
Lewis, S. A., Berg, J. R. & Kleine, T. J. Modulation of epithelial permeability by extracellular macromolecules. Physiol. Rev. 75, 561–589 (1995).
McCartney, F., Gleeson, J. P. & Brayden, D. J. Safety concerns over the use of intestinal permeation enhancers: a mini-review. Tissue Barriers 4, e117682 (2016).
Maher, S., Mrsny, R. J. & Brayden, D. J. Intestinal permeation enhancers for oral peptide delivery. Adv. Drug Deliver. Rev. 106, 277–319 (2016).
Whitehead, K., Karr, N. & Mitragotri, S. Safe and effective permeation enhancers for oral drug delivery. Pharmaceut. Res. 25, 1782–1788 (2008).
Kapitza, C. et al. Oral Insulin: a comparison with subcutaneous regular human insulin in patients with type 2 diabetes. Diabetes Care 33, 1288–1290 (2010).
Banerjee, A. et al. Ionic liquids for oral insulin delivery. Proc. Natl Acad. Sci. USA 115, 7296–7301 (2018).
Iyer, H., Khedkar, A. & Verma, M. Oral insulin – a review of current status. Diabetes Obes. Metab. 12, 179–185 (2010).
Lerner, A. & Matthias, T. Changes in intestinal tight junction permeability associated with industrial food additives explain the rising incidence of autoimmune disease. Autoimmun. Rev. 14, 479–489 (2015).
Pridgen, E. M. et al. Transepithelial transport of Fc-targeted nanoparticles by the neonatal Fc receptor for oral delivery. Sci. Transl. Med. 5, 213ra167 (2013).
Kim, K. S., Suzuki, K., Cho, H., Youn, Y. S. & Bae, Y. H. Oral nanoparticles exhibit specific high-efficiency intestinal uptake and lymphatic transport. ACS Nano. 12, 8893–8900 (2018).
Kim, K. S., Kwag, D. S., Hwang, H. S., Lee, E. S. & Bae, Y. H. Immense Insulin intestinal uptake and lymphatic transport using bile acid conjugated partially uncapped liposome. Mol. Pharmaceut. 15, 4756–4763 (2018).
Cao, Z., Zhang, L. & Jiang, S. Superhydrophilic zwitterionic polymers stabilize liposomes. Langmuir 28, 11625–11632 (2012).
Lu, Y. et al. Micelles with ultralow critical micelle concentration as carriers for drug delivery. Nat. Biomed. Eng. 2, 318–325 (2018).
Thwaites, D. T. & Anderson, C. M. H. The SLC36 family of proton-coupled amino acid transporters and their potential role in drug transport. Brit. J. Pharmacol. 164, 1802–1816 (2011).
Salim, S. Y. & Soderholm, J. D. Importance of disrupted intestinal barrier in inflammatory bowel diseases. Inflamm. Bowel Dis. 17, 362–381 (2011).
Soderholm, J. D. et al. Reversible increase in tight junction permeability to macromolecules in rat ileal mucosa in vitro by sodium caprate, a constituent of milk fat. Digest. Dis. Sci. 43, 1547–1552 (1998).
Brake, R., Vogl, A.W. & Mitchell, A.W.M. Gray’s Anatomy For Students (Elsevier/Churchill Livingstone, 2005).
Frolund, S., Holm, R., Brodin, B. & Nielsen, C. U. The proton-coupled amino acid transporter, SLC36A1 (hPAT1), transports Gly-Gly, Gly-Sar and other Gly-Gly mimetics. Br. J. Pharmacol. 161, 589–600 (2010).
Willems, D., Cadranel, S. & Jacobs, W. Measurement of urinary sugars by HPLC in the estimation of intestinal permeability-evaluation in pediatric clinical-practice. Clin. Chem. 39, 888–890 (1993).
Jiang, X. H., Li, N. & Li, J. S. Intestinal permeability in patients after surgical trauma and effect of enteral nutrition versus parenteral nutrition. World J. Gastroentero. 9, 1878–1880 (2003).
Anderson, C. M. H. et al. H+/amino acid transporter 1 (PAT1) is the imino acid carrier: an intestinal nutrient/drug transporter in human and rat. Gastroenterology 127, 1410–1422 (2004).
Broberg, M. L. et al. Function and expression of the proton-coupled amino acid transporter PAT1 along the rat gastrointestinal tract: implications for intestinal absorption of gaboxadol. Brit. J. Pharmacol. 167, 654–665 (2012).
Olmsted, S. S. et al. Diffusion of macromolecules and virus-like particles in human cervical mucus. Biophys. J. 81, 1930–1937 (2001).
This work was supported by the faculty start-up fund at Wayne State University, the National Science Foundation (grant nos. 1410853 and 1809229) and the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (grant nos. DP2DK111910 and R01DK123293). This work made use of the JEOL 2010 transmission electron microscope supported by National Science Foundation Award No. 0216084. We thank A. Withrow at Michigan State University for supporting tissue sample processing and TEM study. We thank N. Peraino of the Lumigen Instrument Center Mass Spectrometry facilities for access to Shimadzu 8040 (LC–MS–MS). The microscopy and imaging are supported, in part, by NIH Center grant No. P30 CA22453 to the Karmanos Cancer Institute, Wayne State University, and the Perinatology Research Branch of the Nation Institutes of Child Health and Development.
The authors declare no competing interests.
Peer review information Nature Nanotechnology thanks Tim Heise and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 DSPE-PCB/insulin formulations with increasing zinc content showed increased retention of insulin release.
DSPE-PCB/insulin formulations were dialyzed (10 kDa MWCO) against pH 1.2 and 6.8 buffer with 5 mM bile salt at 37 °C. Formulation 1, 2, and 3 had an insulin/ZnCl2 feeding ratio of 50/1, 20/1, and 2.5/1 by weight during the encapsulation process, respectively. Their drug loading is 6.24%, 6.23% and 6.10%, while the corresponding particle sizes are 28.52, 26.36 and 25.96nm respectively. The cumulative released insulin was measured using the BCA assay (N=3 independent experiments, means connected).
Extended Data Fig. 2 Representative images of the tight junction protein ZO-1 of monolayer of Caco-2 cells after treated with different micelles.
The tight junction protein ZO-1 was stained with ZO-1 Monoclonal Antibody Alexa Fluor 488 (green) while the nucleus was stained with Hochest 33342 (blue). Scale bar =20μm. Experiments were repeated three times independently with similar results.
Sodium decanoate caused greater reductions in the TEER of Caco-2 monolayers than polysorbate 80. (N=3 biologically independent samples, means connected). Caco-2 cells were cultured for 96 hours to form monolayers and then treated with different micelles for 4 hours. The electrical resistance was measured at different time points.
Extended Data Fig. 4 Blood glucose profiles for various formulations of DSPE-PCB/insulin capsules on diabetic rats through oral gavage (N=6 biologically independent animals, means connected).
Formulation 0, 1, 2 and 3 had an insulin/ZnCl2 feeding ratio of 75/1, 50/1, 20/1, and 2.5/1 by weight during the encapsulation process, respectively. Their drug loading is 6.25%, 6.24%, 6.23% and 6.10%, while the corresponding particle hydrodynamic sizes are 28.56, 28.52, 26.36 and 25.96 nm respectively.
About this article
Cite this article
Han, X., Lu, Y., Xie, J. et al. Zwitterionic micelles efficiently deliver oral insulin without opening tight junctions. Nat. Nanotechnol. 15, 605–614 (2020). https://doi.org/10.1038/s41565-020-0693-6
Chitosan Coated Luteolin Nanostructured Lipid Carriers: Optimization, In Vitro-Ex Vivo Assessments and Cytotoxicity Study in Breast Cancer Cells
Science Translational Medicine (2021)
Recent advances of nanomedicine-based strategies in diabetes and complications management: Diagnostics, monitoring, and therapeutics
Journal of Controlled Release (2021)
Dealing with the Foreign‐Body Response to Implanted Biomaterials: Strategies and Applications of New Materials
Advanced Functional Materials (2021)